Heterotrimeric G proteins transduce a variety of signals from heptahetical membrane-bound receptors to intracellular effectors. Members of RGS (Regulator of G protein signaling) protein family regulate G protein mediated signals. Members of the Rho family of monomeric GTPases control the organization of the actin cytoskeleton and are involved in a variety of cellular functions. The objective of this proposal is to understand the mechanism of regulation of Rho family GTPases through heterotrimeric G protein-mediated signaling pathways. Among heterotrimeric G proteins, Galpha12 and Galpha13 participate in cellular processes such as cell transformation, neurite retraction, or embryonic development. Rho activation is involved in these signaling pathways. The immediate goal of this proposal is to understand biochemical mechanisms of regulation of the activity of Rho by Galpha12 and Galpha13. Rho family GTPases are regulated by guanine nucleotide exchange factors (GEFs). RhoGEFs with amino terminal RGS domain (RGS-RhoGEFs), p115RhoGEF, LARG, and PDZ-RhoGEF, were identified to function as effectors for Galpha12 and Galpha13. Galpha12/13 interacts with RGS domain of these RhoGEFs and regulates their RhoGEF activity. RGS domains of p115 and LARG act as GTPase activating proteins specific for Galpha12 and Galpha13. The structure basis of the mechanism of regulation of RGS-RhoGEFs by Galpha12 and Galpha13 will be analyzed. The crystal structures of Galpha12 or Galpha13 and its complex with LARG will be solved. The regions of Galpha12/13 or LARG that are involved in their functional interaction will be characterized. The biochemical mechanisms of regulation of RhoGEF activity of LARG or PDZ-RhoGEF will be investigated. Furthermore, Galpha12/13-LARG/PDZRhoGEF signaling pathway in neuronal cells will be analyzed. Galpha12-RhoGEF pathway was identified in C. elegans and its involvement in neuronal function and embryonic development was demonstrated. The physiological significance of this pathway in C. elegans will be investigated using genetical and biochemical methods.